RNA interference or “RNAi” is a term initially coined by Fire and co-workers to describe the observation that double-stranded RNA (dsRNA) can block gene expression when it is introduced into worms (Fire et al., Nature 391:806-811, 1998). Short dsRNA directs gene-specific, post-transcriptional silencing in many organisms, including vertebrates, and has provided a new tool for studying gene function. RNAi has been suggested as a method of developing a new class of therapeutic agents. However, to date, these have remained mostly as suggestions with no demonstrate proof that RNAi can be used therapeutically.
Mammals require molecular oxygen for essential metabolic processes including oxidative phosphorylation in which oxygen serves as electron acceptor during ATP formation. Systemic, local, and intracellular homeostatic responses elicited by hypoxia (the state in which oxygen demand exceeds supply) include erythropoiesis by individuals who are anemic or at high altitude (Jelkmann (1992) Physiol. Rev. 72:449-489), neovascularization in ischemic myocardium (White et al. (1992) Circ. Res. 71:1490-1500), and glycolysis in cells cultured at reduced oxygen tension (Wolfle et al. (1983) Eur. J. Biochem. 135:405-412). These adaptive responses either increase oxygen delivery or activate alternate metabolic pathways that do not require oxygen. Hypoxia-inducible gene products that participate in these responses include erythropoietin (EPO) (reviewed in Semenza (1994) Hematol. Oncol. Clinics N. Amer. 8:863-884), vascular endothelial growth factor (Shweiki et al. (1992) Nature 359:843-845; Banai et al. (1994) Cardiovasc. Res. 28:1176-1179; Goldberg & Schneider (1994) J. Biol. Chem. 269:4355-4359), and glycolytic enzymes (Firth et al. (1994) Proc. Natl. Acad. Sci. USA 91:6496-6500; Semenza et al. (1994) J. Biol. Chem. 269:23757-23763).
The molecular mechanisms that mediate genetic responses to hypoxia have been extensively investigated for the EPO gene, which encodes a growth factor that regulates erythropoiesis and thus blood oxygen carrying capacity (Jelkmann (1992) supra; Semenza (1994) supra). Cis-acting DNA sequences required for transcriptional activation in response to hypoxia were identified in the EPO 3′-flanking region and a trans-acting factor that binds to the enhancer, hypoxia-inducible factor 1 (HIF-1), fulfilled criteria for a physiological regulator of EPO transcription. Inducers of EPO expression (1% oxygen, cobalt chloride [CoCl2], and desferrioxamine [DFX]) also induced HIF-1 DNA binding activity with similar kinetics; inhibitors of EPO expression (actinomycin D, cycloheximide, and 2-aminopurine) blocked induction of HIF-1 activity; and mutations in the EPO 3′-flanking region that eliminated HIF-1 binding also eliminated enhancer function (Semenza (1994) supra). These results also support the hypothesis that oxygen tension is sensed by a hemoprotein (Goldberg et al. (1988) Science 242:1412-1415) and that a signal transduction pathway requiring ongoing transcription, translation, and protein phosphorylation participates in the induction of HIF-1 DNA-binding activity and EPO transcription in hypoxic cells (Semenza (1994) supra).
EPO expression is cell type specific, but induction of HIF-1α activity by 1% oxygen, CoCl2, or DFX was detected in many mammalian cell lines (Wang & Semenza (1993a) Proc. Natl. Acad. Sci. USA 90:4304-4308), and the EPO enhancer directed hypoxia-inducible transcription of reporter genes transfected into non-EPO-producing cells (Wang & Semenza (1993a) supra; Maxwell et al. (1993) Proc. Natl. Acad. Sci. USA 90:2423-2427). RNAs encoding several glycolytic enzymes were induced by 1% oxygen, CoCl.sub.2, or DFX in EPO-producing Hep3B or non-producing HeLa cells whereas cycloheximide blocked their induction and glycolytic gene sequences containing HIF-1 binding sites mediated hypoxia-inducible transcription in transfection assays (Firth et al. (1994) supra; Semenza et al. (1994) supra). These experiments support the role of HIF-1 in activating homeostatic responses to hypoxia.
HIF-1 is a dimer composed of HIF-1α and HIF-1β subunits. While the HIF-1β subunit is constitutively expressed, the HIF-1α subunit is the limiting member of the heterodimer and therefore regulates HIF-1 levels. Under normoxic conditions, HIF-1α is ubiquinated and rapidly degraded. However, under hypoxic conditions the rate of ubiquitination dramatically decreases and HIF-1α is stabilized, resulting in upregulation of HIF-1 dimer. This is an important point and provides the rationale for targeting HIF-1α instead of HIF-1β for modulating HIF-1 activity.
Macular degeneration is a major cause of blindness in the United States and the frequency of this disorder increases with age. Macular degeneration refers to the group of diseases in which sight-sensing cells in the macular zone of the retina malfunction or loose function and which can result in debilitating loss of vital central or detail vision.
Age-related macular degeneration (AMD), which is the most common form of macular degeneration, occurs in two main forms. Ninety percent of people with AMD have the form described as “dry” macular degeneration. An area of the retina is affected, which leads to slow breakdown of cells in the macula, and a gradual loss of central vision. The other form of AMD is “wet” macular degeneration. Although only 10% of people with AMD have this type, it accounts for 90% of blindness from the disease. As dry AMD progresses, new blood vessels may begin to grow and cause “wet” AMD. These new blood vessels often leak blood and fluid under the macula. This causes rapid damage to the macula that can lead to loss of central vision in a short time. iRNA agents targeting HIF-1α can be useful for the treatment of wet and dry macular degeneration.